Mycobacterium tuberculosis is a leading AIDS-related infectious disease killer worldwide. Tuberculosis is a disease of multiple pathologic stages, and hence M. tb. must possess multiple adaptive genetic strategies to survive in these differing environments. Understanding the organism's pathogenesis mechanisms during these disease stages is the surest way to develop better diagnostics, vaccines, and drugs which are critically needed for patients with TB and TB/HIV. We have genetically interrupted 5 M. tb. sigma factor genes, identified the genes which they regulate by using microarray technology and assessed their virulence in mice. Among these alternative sigma factors there is functional redundancy in that upon infection of mice 4 of the 5 mutants show the immunopathology phenotype of attenuation in which mycobacterial counts are maintained at high level but there is a significant delay in mortality and in disease progression in the lungs. In the first aim, we will explore the immunopathology defect demonstrated by several of the sigma factor knockout mutants. The roles of known mediators of TB control such as nitric oxide, TNF-alpha, interferon-gamma and phagocyte oxidase will be examined using the M. tb. deltasigH and other knockout mutants which display the immunopathology phenotype. Second, we will refine our understanding of these sigma factor regulons by studying expression profiles under stress conditions, by biochemical analysis of transcription, by constructing double knockout mutants, and through conditional expression of sigma factors. We will address whether there is sufficient ECF promoter consensus degeneracy to permit redundant sigma factor control of dependent genes or whether there are distal mediators which remain to be discovered. Finally, in the 3rd aim we will explore the modulation of sigma factor activity by studying anti-sigma factors. We will study the effect of AsiA, a phage-encoded anti-sigma factor which binds to RNA polymerase, remodels it, and alters its promoter specificity. We will seek to determine whether AsiA or portions of it have a transcription-specificity modifying effect in mycobacteria. We will also study the role of a novel sigma factor-regulator in M. tb, Rv1364c, which our data show is required for resistance to SDS stress. Advancing our understanding ofM. tb sigma factors and their related regulators will help establish key adaptive mechanisms in the pathogenesis of TB.